Inbred corn plant 94INK1A and seeds thereof

ABSTRACT

According to the invention, there is provided an inbred corn plant designated 94INK1A. This invention thus relates to the plants, seeds and tissue cultures of the inbred corn plant 94INK1A, and to methods for producing a corn plant produced by crossing the inbred corn plant 94INK1A with itself or with another corn plant, such as another inbred. This invention further relates to corn seeds and plants produced by crossing the inbred plant 94INK1A with another corn plant, such as another inbred, and to crosses with related species. This invention further relates to the inbred and hybrid genetic complements of the inbred corn plant 94INK1A, and also to the SSR and genetic isozyme typing profiles of inbred corn plant 94INK1A.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to inbred corn seed and plantsdesignated 94INK1A, and derivatives and tissue cultures thereof.

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection: S₁→S₂; S₂→S₃; S₃→S₄; S4S5, etc. After at least fivegenerations, the inbred plant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. The hybrid progeny of the first generation is designated F₁.Typically, F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, is manifested in many polygenic traits,including markedly improved yields, better stalks, better roots, betteruniformity and better insect and disease resistance. In the developmentof hybrids only the F₁ hybrid plants are typically sought. An F₁ singlecross hybrid is produced when two inbred plants are crossed. A doublecross hybrid is produced from four inbred plants crossed in pairs (A×Band C×D) and then the two F₁ hybrids are crossed again (A×B)×(C×D).

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant designated94INK1A. Also provided are corn plants having all the physiological andmorphological characteristics of corn plant 94INK1A. The inbred cornplant of the invention may further comprise, or have, a cytoplasmic ornuclear factor that is capable of conferring male sterility. Parts ofthe corn plant of the present invention are also provided, for example,pollen obtained from an inbred plant and an ovule of the inbred plant.

The invention also concerns seed of the corn plant 94INK1A. A sample ofthis seed has been deposited under ATCC Accession No. PTA-2119. Theinbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed of the corn plant designated94INK1A. Essentially homogeneous populations of inbred seed are thosethat consist essentially of the particular inbred seed, and aregenerally free from substantial numbers of other seed, so that theinbred seed forms between about 90% and about 100% of the total seed,and preferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 99.9% of inbredseed, as measured by seed grow outs.

Therefore, in the practice of the present invention, inbred seedgenerally forms at least about 97% of the total seed. However, even if apopulation of inbred corn seed was found, for some reason, to containabout 50%, or even about 20% or 15% of inbred seed, this would still bedistinguished from the small fraction of inbred seed that may be foundwithin a population of hybrid seed, e.g., within a bag of hybrid seed.In such a bag of hybrid seed offered for sale, the Governmentalregulations require that the hybrid seed be at least about 95% of thetotal seed. In the most preferred practice of the invention, the femaleinbred seed that may be found within a bag of hybrid seed will be about1% of the total seed, or less, and the male inbred seed that may befound within a bag of hybrid seed will be negligible, i.e., will be onthe order of about a maximum of 1 per 100,000, and usually less thanthis value.

The population of inbred corn seed of the invention can further beparticularly defined as being essentially free from hybrid seed. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of inbred corn plants designated 94INK1A.

In another aspect of the invention, single locus converted plants of94INK1A are provided. The single transferred locus may preferably be adominant or recessive allele. Preferably, the single transferred locuswill confer such traits as male sterility, yield stability, waxy starch,yield enhancement, industrial usage, herbicide resistance, insectresistance, resistance to bacterial, fungal, nematode or viral disease,male fertility, and enhanced nutritional quality. The single locus maybe a naturally occurring maize gene or a transgene introduced throughgenetic transformation techniques. When introduced throughtransformation, a single locus may comprise one or more transgenesintegrated at a single chromosomal location.

In yet another aspect of the invention, an inbred corn plant designated94INK1A is provided, wherein a cytoplasmically-inherited trait has beenintroduced into said inbred plant. Such cytoplasmically-inherited traitsare passed to progeny through the female parent in a particular cross.An exemplary cytoplasmically-inherited trait is the male sterilitytrait. A cytoplasmically inherited trait may be a naturally occurringmaize trait or a trait introduced through genetic transformationtechniques.

In another aspect of the invention, a tissue culture of regenerablecells of inbred corn plant 94INK1A is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the foregoinginbred corn plant, and of regenerating plants having substantially thesame genotype as the foregoing inbred corn plant. Examples of some ofthe physiological and morphological characteristics of the inbred cornplant 94INK1A include characteristics related to yield, maturity, andkernel quality, each of which are specifically disclosed herein. Theregenerable cells in such tissue cultures will preferably be derivedfrom embryos, meristematic cells, immature tassels, microspores, pollen,leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs,husks, or stalks, or callus or protoplasts derived from these tissues.Still further, the present invention provides corn plants regeneratedfrom the tissue cultures of the invention, the plants having all thephysiological and morphological characteristics of corn plant 94INK1A.

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is theinbred corn plant designated 94INK1A. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second, distinctinbred corn plant to provide a hybrid that has, as one of its parents,the inbred corn plant 94INK1A. In these processes, the step of crossingwill result in the production of seed. The seed production occursregardless of whether the seed is collected or not.

In a preferred embodiment of the invention, crossing comprises plantingin pollinating proximity seeds of a first and second parent corn plant,and preferably, seeds of a first inbred corn plant and a second,distinct inbred corn plant; cultivating or growing the seeds of saidfirst and second parent corn plants into plants that bear flowers;emasculating the male flowers of the first or second parent corn plant,(i.e., treating or manipulating the flowers so as to prevent pollenproduction, in order to produce an emasculated parent corn plant)allowing natural cross-pollination to occur between the first and secondparent corn plants; and harvesting the seeds from the emasculated parentcorn plant. Where desired, the harvested seed is grown to produce a cornplant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is the inbred corn plant designated 94INK1A. In oneembodiment of the invention, corn plants produced by the process arefirst generation (F₁) hybrid corn plants produced by crossing an inbredin accordance with the invention with another, distinct inbred. Thepresent invention further contemplates seed of an F₁ hybrid corn plant.Therefore, certain exemplary embodiments of the invention provide an F₁hybrid corn plant and seed thereof. An example of such a hybrid whichcan be produced with the inbred designated 94INK1A is the hybrid cornplant designated 8002697.

In still yet another aspect of the invention, an inbred geneticcomplement of the corn plant designated 94INK1A is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a corn plant, or a cell or tissue of that plant. Aninbred genetic complement thus represents the genetic make up of aninbred cell, tissue or plant, and a hybrid genetic complement representsthe genetic make up of a hybrid cell, tissue or plant. The inventionthus provides corn plant cells that have a genetic complement inaccordance with the inbred corn plant cells disclosed herein, andplants, seeds and diploid plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.Thus, such corn plant cells may be defined as having an SSR geneticmarker profile in accordance with the profile shown in Table 4, or agenetic isozyme typing profile in accordance with the profile shown inTable 5, or having both an SSR genetic marker profile and a geneticisozyme typing profile in accordance with the profiles shown in Table 4and Table 5. It is understood that 94INK1A could also be identified byother types of genetic markers such as, for example, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS OF PLANTCHARACTERISTICS

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks. Minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of intemodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units which are calculated by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for an inbred line or hybrid to have approximately 50% of theplants shedding pollen as measured from time of planting. GDUs to shedis determined by summing the individual GDU daily values from plantingdate to the date of 50% pollen shed.

GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50% of the plants with silk emergence asmeasured from time of planting. GDUs to silk is determined by summingthe individual GDU daily values from planting date to the date of 50%silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. +=indicates the presence of Htchlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−=indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or lax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination. Rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. OTHER DEFINITIONS

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Electrophoresis: A process by which particles suspended in a fluid or agel matrix are moved under the action of an electrical field, andthereby separated according to their charge and molecular weight. Thismethod of separation is well known to those skilled in the art and istypically applied to separating various forms of enzymes and of DNAfragments produced by restriction endonucleases.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes: Detectable variants of an enzyme, the variants catalyzing thesame reaction(s) but differing from each other, e.g., in primarystructure and/or electrophoretic mobility. The differences betweenisozymes are under single gene, codorninant control. Consequently,electrophoretic separation to produce band patterns can be equated todifferent alleles at the DNA level. Structural differences that do notalter charge cannot be detected by this method.

Isozyme typing profile: A profile of band patterns of isozymes separatedby electrophoresis that can be equated to different alleles at the DNAlevel.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

94INK1A: The corn plant from which seeds having ATCC Accession No.PTA-2119 were obtained, as well as plants grown from those seeds.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

SSR genetic marker profile: A profile of simple sequence repeats scoredby gel electrophoresis following PCR™ amplification using flankingoligonucleotide primers.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the desired morphological and physiological characteristics of aninbred are recovered in addition to the characteristics conferred by thesingle locus transferred into the inbred via the backcrossing technique.A single locus may comprise one gene, or in the case of transgenicplants, one or more transgenes integrated into the host genome at asingle site (locus).

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence which has been introduced into the nuclearor chloroplast genome of a maize plant by a genetic transformationtechnique.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

III. INBRED CORN PLANT 94INK1A

In accordance with one aspect of the present invention, there isprovided a novel inbred corn plant, designated 94INK1A. A description ofinbred corn plant 94INK1A is provided herein below.

A. Origin and Breeding History

Inbred plant 94INK1A was derived from the cross between the lines 3IIH6and 94KBZ1. The origin and breeding history of inbred plant 94INK1A canbe summarized as follows:

Summer The inbred line 3IIH6 (a proprietary DEKALB Genetics 1992Corporation inbred) was crossed to the inbred line 94KBZ1 (a proprietaryDEKALB Genetics Corporation inbred) in nursery row G92: 1-2 andG92:3-12. Winter The S0 seed was grown and self-pollinated in nursery1992-93 row C92:1936. Summer The S1 seed was grown and self-pollinatedin nursery 1993 rows G93:19-98 through 19-100 and 20-100 through 20-54.Thirty-three ears were selected. Summer S2 ears were grown ear-to-rowand self-pollinated. Two ears 1994 were selected in nursery rowG94:59-71. Summer S3 ears were grown ear-to-row and self-pollinated.Three ears 1995 were selected in nursery row G95:64-65. Summer S4 earswere grown ear-to-row and self-pollinated. Four ears 1996 were selectedin nursery row G96:401-78. Summer S5 ears were grown ear-to-row andself-pollinated. Two ears 1997 from nursery row G97:502-70 were selectedand designated as coded inbred 94INK1A. Winter S6 ears were grownear-to-row and self-pollinated. Twenty 1997-98 ears from nursery rowsH97 1 1Y:3-23 through 3-26 were selected. Summer S7 ears were grownear-to-row and self-pollinated. Fifteen 1998 ears were selected fromnursery row G98:35-83 through 35-89. Winter S8 ears were grownear-to-row and self-pollinated. Final 1998-99 selection was completed innursery rows PR98:7-1 through 7-17. This selection consisted of bulkingS9 ears.

94INK1A shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 1.94INK1A has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in 94INK1A.

Inbred corn plants can be reproduced by planting the seeds of the inbredcorn plant 94INK1A, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant 94INK1A. A description of thephysiological and morphological characteristics of corn plant 94INK1Aand comparative inbreds is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics for the 94INK1APhenotype VALUE CHARACTERISTIC 94INK1A 3IIH6 94KBZ1 94INK1B 1. STALKDiameter (width) cm. 2.2 2.5 2.0 2.2 Brace Root Color Moderate Absent −Moderate Anthocyanin Absent Absent Absent Basal-Weak Nodes With Brace0.8 1.3 1.3 0.9 Roots Internode Direction Straight Straight StraightStraight Internode Length cm. 14.3 13.9 15.2 14.8 2. LEAF Color GreenGreen Green Green Length cm. 68.4 36.2 46.4 66.5 Width cm. 9.1 4.3 6.79.3 Sheath Anthocyanin Basal-Weak — Absent to Basal-Weak Basal-WeakSheath Pubescence Light Moderate Light Light Marginal Waves Few Few FewFew Longitudinal Creases Moderate Moderate Moderate Few 3. TASSEL Lengthcm. 38.5 41.0 38.3 43.1 Spike Length cm. 20.6 24.4 19.1 21.1 PeduncleLength cm. 10.5 7.5 9.3 9.7 Branch Number 5.0 13.1 8.0 5.3 Anther ColorGreen-Yellow Red Green-Yellow Green-Yellow Glume Color Green Green GreenGreen Glume Band Absent Absent Absent Absent 4. EAR Silk Color Pink RedRed Green-Yellow Number Per Stalk 2.0 1.2 1.1 1.0 Position (attitude)Pendant — − Pendant Length cm. 14.1 15.8 14.0 14.0 Shape Semi-ConicalSemi-Conical Semi-Conical Semi-Conical Diameter cm. 3.7 3.7 3.3 3.8Weight gm. − 103.1 72.9 — Shank Length cm. 13.1 12.2 12.3 11.0 HuskBract Short Short Short Short Husk Cover cm. 7.0 3.4 2.6 5.4 Husk ColorFresh Green Green Green Green Husk Color Dry Buff Buff Buff Buff HuskOpening Very Loose Moderate Moderate Very Loose Cob Diameter cm. 2.2 2.11.8 2.2 Cob Color Red Red Pink Red Shelling Percent 84.8 85.3 86.5 83.95. KERNEL Row Number 14.8 15.7 13.2 14.4 Number Per Row 28.8 30.1 27.023.6 Type Semi-Dent Dent Dent Semi-Dent Row Direction Straight SlightlyCurved Slightly Curved Straight Cap Color Orange Yellow Yellow YellowSide Color Orange Deep-Yellow Orange Yellow Length (depth) mm. 9.8 10.19.7 10.0 Width mm. 9.4 7.4 7.5 8.6 Thickness 4.0 4.5 4.5 3.8 Weight of1000K gm. 259.0 223.1 220.6 291.0 Endosperm Type Normal Normal NormalNormal Endosperm Color Yellow Yellow Yellow Yellow

C. Deposit Information

A deposit of 2500 seeds of the inbred corn plant designated 94INK1A hasbeen made with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. on Jun. 21, 2000. Those deposited seedshave been assigned ATCC Accession No. PTA-2119. The deposit was made inaccordance with the terms and provisions of the Budapest Treaty relatingto deposit of microorganisms and was made for a term of at least thirty(30) years and at least five (05) years after the most recent requestfor the furnishing of a sample of the deposit is received by thedepository, or for the effective term of the patent, whichever islonger, and will be replaced if it becomes non-viable during thatperiod.

IV. SINGLE LOCUS CONVERSIONS

When the term inbred corn plant is used in the context of the presentinvention, this also includes any single locus conversions of thatinbred. The term single locus converted plant as used herein refers tothose corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single locus transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the locus or loci for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original inbred of interest (recurrent parent) is crossed to asecond inbred (nonrecurrent parent) that carries the single locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred locus from the nonrecurrent parent. The backcross processmay be accelerated by the use of genetic markers, such as SSR, RFLP, SNPor AFLP markers to identify plants with the greatest genetic complementfrom the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single locus of the recurrent inbred ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus, but may be inherited through the cytoplasm. Some knownexceptions to this are genes for male sterility, some of which areinherited cytoplasmically, but still act as single locus traits. Anumber of exemplary single locus traits are described in, for example,PCT Application WO 95/06128, the disclosure of which is specificallyincorporated herein by reference.

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. A particularly useful type of malesterility gene is one which can be induced by exposure to a chemicalagent, for example, a herbicide (U.S. patent application Ser. No.08/927,368, filed Sep. 11, 1997, the disclosure of which is specificallyincorporated herein by reference in its entirety). Both inducible andnon-inducible male sterility genes can increase the efficiency withwhich hybrids are made, in that they eliminate the need to physicallyemasculate the corn plant used as a female in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns the inbred corn plant 94INK1A comprising a single gene capableof restoring male fertility in an otherwise male-sterile inbred orhybrid plant. Examples of male-sterility genes and correspondingrestorers which could be employed with the inbred of the invention arewell known to those of skill in the art of plant breeding and aredisclosed in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat. No.5,689,041; U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

Direct selection may be applied where a single locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful single locus traits are those which are introduced bygenetic transformation techniques. Methods for the genetictransformation of maize are known to those of skill in the art. Forexample, methods which have been described for the genetictransformation of maize include electroporation (U.S. Pat. No.5,384,253), electrotransformation (U.S. Pat. No. 5,371,003),microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No.5,736,369, U.S. Pat. No. 5,538,880; and PCT Publication WO 95/06128),Agrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 and E.P.Publication EP672752), direct DNA uptake transformation of protoplasts(Omirulleh et al., 1993) and silicon carbide fiber-mediatedtransformation (U.S. Pat. No. 5,302,532 and U.S. Pat. No. 5,464,765).

A type of single locus trait which can be introduced by genetictransformation (U.S. Pat. No. 5,554,798) and has particular utility is agene which confers resistance to the herbicide glyphosate. Glyphosateinhibits the action of the enzyme EPSPS, which is active in thebiosynthetic pathway of aromatic amino acids. Inhibition of this enzymeleads to starvation for the amino acids phenylalanine, tyrosine, andtryptophan and secondary metabolites derived therefrom. Mutants of thisenzyme are available which are resistant to glyphosate. For example,U.S. Pat. No. 4,535,060 describes the isolation of EPSPS mutations whichconfer glyphosate resistance upon organisms having the Salmonellatyphimurium gene for EPSPS, aroA. A mutant EPSPS gene having similarmutations has also been cloned from Zea mays. The mutant gene encodes aprotein with amino acid changes at residues 102 and 106 (PCT PublicationWO 97/04103). When a plant comprises such a gene, a herbicide resistantphenotype results.

Plants having inherited a transgene comprising a mutated EPSPS gene may,therefore, be directly treated with the herbicide glyphosate without theresult of significant damage to the plant. This phenotype providesfarmers with the benefit of controlling weed growth in a field of plantshaving the herbicide resistance trait by application of the broadspectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields whereglyphosate resistant corn plants are grown. The herbicide applicationrates may typically range from 4 ounces of ROUNDUP™ to 256 ouncesROUNDUP™ per acre. More preferably, about 16 ounces to about 64 ouncesper acre of ROUNDUP™ may be applied to the field. However, theapplication rate may be increased or decreased as needed, based on theabundance and/or type of weeds being treated. Additionally, depending onthe location of the field and weather conditions, which will influenceweed growth and the type of weed infestation, it may be desirable toconduct further glyphosate treatments. The second glyphosate applicationwill also typically comprise an application rate of about 16 ounces toabout 64 ounces of ROUNDUP™ per acre treated. Again, the treatment ratemay be adjusted based on field conditions. Such methods of applicationof herbicides to agricultural crops are well known in the art and aresummarized in general in Anderson (1983).

Alternatively, more than one single locus trait may be introgressed intoan elite inbred by the method of backcross conversion. A selectablemarker gene and a gene encoding a protein which confers a trait ofinterest may be simultaneously introduced into a maize plant as a resultof genetic transformation. Usually one or more introduced genes willintegrate into a single chromosome site in the host cell's genome. Forexample, a selectable marker gene encoding phosphinothricin acetyltransferase (PPT) (e.g., a bar gene) and conferring resistance to theactive ingredient in some herbicides by inhibiting glutamine synthetase,and a gene encoding an endotoxin from Bacillus thuringiensis (Bt) andconferring resistance to particular classes of insects, e.g.,lepidopteran insects, in particular the European Corn Borer, may besimultaneously introduced into a host genome. Furthermore, through theprocess of backcross conversion more than one transgenic trait may betransferred into an elite inbred.

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

V. ORIGIN AND BREEDING HISTORY OF AN EXEMPLARY SINGLE LOCUS CONVERTEDPLANT

85DGD1 MLms is a single locus conversion of 85DGD1 to cytoplasmic malesterility. 85DGD1 MLms was derived using backcross methods. 85DGD1 (aproprietary inbred of DEKALB Genetics Corporation) was used as therecurrent parent and MLms, a germplasm source carrying ML cytoplasmicsterility, was used as the nonrecurrent parent. The breeding history ofthe single locus converted inbred 85DGD1 MLms can be summarized asfollows:

Hawaii Nurseries Planting Date Apr. 2, 1992 Made up S-O: Female row 585male row 500

Hawaii Nurseries Planting Date Jul. 15, 1992 S-O was grown and plantswere backcrossed times 85DGD1 (rows 444′ 443)

Hawaii Nurseries Planting Date Nov. 18, 1992 Bulked seed of the BC1 wasgrown and backcrossed times 85DGD1 (rows V3-27′ V3-26)

Hawaii Nurseries Planting Date Apr. 2, 1993 Bulked seed of the BC2 wasgrown and backcrossed times 85DGD1 (rows 37′ 36)

Hawaii Nurseries Planting Date Jul. 14, 1993 Bulked seed of the BC3 wasgrown and backcrossed times 85DGD1. (rows 99′ 98)

Hawaii Nurseries Planting Date Oct. 28, 1993 Bulked seed of BC4 wasgrown and backcrossed times 85DGD1 (rows KS-63′ KS-62)

Summer 1994 A single ear of the BC5 was grown and backcrossed times85DGD1 (MC94-822′ MC94-822-7)

Winter 1994 Bulked seed of the BC6 was grown and backcrossed times85DGD1 (3Q-1′ 3Q-2)

Summer 1995 Seed of the BC7 was bulked and named 85DGD1 MLms.

VI. TISSUE CULTURES AND IN VITRO REGENERATION OF CORN PLANTS

A further aspect of the invention relates to tissue cultures of the cornplant designated 94INK1A. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

VII. TASSEL/ANTHER CULTURE

Tassels contain anthers which in turn enclose microspores. Microsporesdevelop into pollen. For anther/microspore culture, if tassels are theplant composition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., 1987), trypan blue (preferred) and acetocarmine squashing. Themid-uninucleate microspore stage has been found to be the developmentalstage most responsive to the subsequent methods disclosed to ultimatelyproduce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

In embodiments where microspores are obtained from anthers, microsporescan be released from the anthers into an isolation medium following themannitol preculture step. One method of rel+ease is by disruption of theanthers, for example, by chopping the anthers into pieces with a sharpinstrument, such as a razor blade, scalpel, or Waring blender. Theresulting mixture of released microspores, anther fragments, andisolation medium are then passed through a filter to separatemicrospores from anther wall fragments. An embodiment of a filter is amesh, more specifically, a nylon mesh of about 112 mm pore size. Thefiltrate which results from filtering the microspore-containing solutionis preferably relatively free of anther fragments, cell walls, and otherdebris.

In a preferred embodiment, isolation of microspores is accomplished at atemperature below about 25° C. and preferably, at a temperature of lessthan about 15° C. Preferably, the isolation media, dispersing tool(e.g., razor blade), funnels, centrifuge tubes, and dispersing container(e.g., petri dish) are all maintained at the reduced temperature duringisolation. The use of a precooled dispersing tool to isolate maizemicrospores has been reported (Gaillard et al., 1991).

Where appropriate and desired, the anther filtrate is then washedseveral times in isolation medium. The purpose of the washing andcentrifugation is to eliminate any toxic compounds which are containedin the non-microspore part of the filtrate and are created by thechopping process. The centrifugation is usually done at decreasing spinspeeds, for example, 1000, 750, and finally 500 rpms. The result of theforegoing steps is the preparation of a relatively pure tissue culturesuspension of microspores that are relatively free of debris and antherremnants.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. The microsporesuspension is layered onto a support, for example by pipetting. Thereare several types of supports which are suitable and are within thescope of the invention. An illustrative embodiment of a solid support isa TRANSWELL® culture dish. Another embodiment of a solid support fordevelopment of the microspores is a bilayer plate wherein liquid mediais on top of a solid base. Other embodiments include a mesh or amillipore filter. Preferably, a solid support is a nylon mesh in theshape of a raft. A raft is defined as an approximately circular supportmaterial which is capable of floating slightly above the bottom of atissue culture vessel, for example, a petri dish, of about a 60 or 100mm size, although any other laboratory tissue culture vessel willsuffice. In an illustrative embodiment, a raft is about 55 mm indiameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent). The charcoal is believed to absorb toxic wastes andintermediaries. The solid medium allows embryoids to mature.

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium. An advantage of the raft is to permit diffusion of nutrients tothe microspores. Use of a raft also permits transfer of the microsporesfrom dish to dish during subsequent subculture with minimal loss,disruption, or disturbance of the induced embryoids that are developing.The rafts represent an advantage over the multi-welled TRANSWELL®plates, which are commercially available from COSTAR, in that thecommercial plates are expensive. Another disadvantage of these plates isthat to achieve the serial transfer of microspores to subsequent media,the membrane support with cells must be peeled off the insert in thewells. This procedure does not produce as good a yield nor as efficienttransfers, as when a mesh is used as a vehicle for cell transfer.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

After the microspores have been isolated, they are cultured in a lowstrength anther culture medium until about the 50 cell stage when theyare subcultured onto an embryoid/callus maturation medium. Medium isdefined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, and growth regulators. A solidifying agent isoptional. A preferred embodiment of such a media is referred to as “Dmedium,” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

In an illustrative embodiment, 1 ml of isolated microspores are pipettedonto a 10 mm nylon raft and the raft is floated on 1.2 ml of medium “D,”containing sucrose or preferably maltose. Both calli and embryoids candevelop. Calli are undifferentiated aggregates of cells. Type I is arelatively compact, organized, and slow growing callus. Type II is asoft, friable, and fast-growing one. Embryoids are aggregates exhibitingsome embryo-like structures. The embryoids are preferred for subsequentsteps to regenerating plants. Culture medium “D” is an embodiment ofmedium that follows the isolation medium and replaces it. Medium “D”promotes growth to an embryoid/callus. This medium comprises 6N1 saltsat ⅛ the strength of a basic stock solution (major components) and minorcomponents, plus 12% sucrose, or preferably 12% maltose, 0.1 mg/l B1,0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5 mg/l silver nitrate.Silver nitrate is believed to act as an inhibitor to the action ofethylene. Multi-cellular structures of approximately 50 cells eachgenerally arise during a period of 12 days to 3 weeks. Serial transferafter a two week incubation period is preferred.

After the petri dish has been incubated for an appropriate period oftime, preferably two weeks in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media which promote embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12% sugar(sucrose, maltose, or a combination thereof), 0.5% activated charcoal,400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percentGELRITE™ (solidifying agent) and is capable of promoting the maturationof the embryoids. Higher quality embryoids, that is, embryoids whichexhibit more organized development, such as better shoot meristemformation without precocious germination, were typically obtained withthe transfer to full strength medium compared to those resulting fromcontinuous culture using only, for example, the isolated microsporeculture (IMC) Medium “D.” The maturation process permits the pollenembryoids to develop further in route toward the eventual regenerationof plants. Serial transfer occurs to full strength solidified 6N1 mediumusing either the nylon raft, the TRANSWELL® membrane, or bilayer plates,each one requiring the movement of developing embryoids to permitfurther development into physiologically more mature structures. In anespecially preferred embodiment, microspores are isolated in anisolation media comprising about 6% maltose, cultured for about twoweeks in an embryoid/calli induction medium comprising about 12% maltoseand then transferred to a solid medium comprising about 12% sucrose.

At the point of transfer of the raft, after about two weeks ofincubation, embryoids exist on a nylon support. The purpose oftransferring the raft with the embryoids to a solidified medium afterthe incubation is to facilitate embryo maturation. Mature embryoids atthis point are selected by visual inspection indicated by zygoticembryo-like dimensions and structures and are transferred to the shootinitiation medium. It is referred that shoots develop before roots, orthat shoots and roots develop concurrently. roots develop before shoots,plant regeneration can be impaired. To produce solidified media, thebottom of a petai dish of approximately 100 nun is covered with about 30ml of 0.2% GELRITE™ solidified medium. A sequence of regeneration mediaare used for whole plant formation from the embryoids.

During the regeneration process, individual embryoids are induced toform plantlets. The number of different media in the sequence can varydepending on the specific protocol used. Finally, a rooting medium isused as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

Plants have been produced from isolated microspore cultures by themethods disclosed herein, including self-pollinated plants. The rate ofembryoid induction was much higher with the synergistic preculturetreatment consisting of a combination of stress factors, including acarbon source which can be capable of inducing starvation, a coldtemperature, and colchicine, than has previously been reported. Anillustrative embodiment of the synergistic combination of treatmentsleading to the dramatically improved response rate compared to priormethods, is a temperature of about 10° C., mannitol as a carbon source,and 0.05% colchicine.

The inclusion of ascorbic acid, an anti-oxidant, in the isolation mediumis preferred for maintaining good microspore viability. However, thereseems to be no advantage to including mineral salts in the isolationmedium. The osmotic potential of the isolation medium was maintainedoptimally with about 6% sucrose, although a range of 2% to 12% is withinthe s cope of this invention.

In an embodiment of the embryoid/callus organizing media, mineral saltsconcentration in IMC Culture Media “D” is (⅛×), the concentration whichis used also in anther culture medium. The 6N1 salts major componentshave been modified to remove ammonium nitrogen. Osmotic potential in theculture medium is maintained with about 12% sucrose and about 400 mg/lproline. Silver nitrate (0.5 mg/l) was included in the medium to modifyethylene activity. The preculture media is further characterized byhaving a pH of about 5.7 to 6.0. Silver nitrate and vitamins do notappear to be crucial to this medium but do improve the efficiency of theresponse.

Whole anther cultures can also be used in the production ofmonocotyledonous plants from a plant culture system. There are somebasic similarities of anther culture methods and microspore culturemethods with regard to the media used. A difference from isolatedmicrospore cultures is that undisrupted anthers are cultured, so that asupport, e.g., a nylon mesh support, is not needed. The first step indeveloping the anther cultures is to incubate tassels at a coldtemperature. A cold temperature is defined as less than about 25° C.More specifically, the incubation of the tassels is preferably performedat about 10° C. A range of 8 to 14° C. is also within the scope of theinvention. The anthers are then dissected from the tassels, preferablyafter surface sterilization using forceps, and placed on solidifiedmedium. An example of such a medium is designated 6N1-TGR-P4.

The anthers are then treated with environmental conditions that arecombinations of stresses that are capable of diverting microspores fromgametogenesis to embryogenesis. It is believed that the stress effect ofsugar alcohols in the preculture medium, for example, mannitol, isproduced by inducing starvation at the predefined temperature. In oneembodiment, the incubation pretreatment is for about 14 days at 10° C.It was found that treating the anthers in addition with a carbonstructure, an illustrative embodiment being a sugar alcohol, preferablymannitol, produces dramatically higher anther culture response rates asmeasured by the number of eventually regenerated plants, than bytreatment with either cold treatment or mannitol alone. These resultsare particularly surprising in light of teachings that cold is betterthan mannitol for these purposes, and that warmer temperatures interactwith mannitol better.

To incubate the anthers, they are floated on a preculture medium whichdiverts the microspores from gametogenesis, preferably on a mannitolcarbon structure, more specifically, 0.3 M of mannitol plus 50 mg/l ofascorbic acid. Three milliliters is about the total amount in a dish,for example, a tissue culture dish, more specifically, a 60 mm petridish. Anthers are isolated from about 120 spikelets for one dish yieldsabout 360 anthers.

Chromosome doubling agents can be used in the preculture media foranther cultures. Several techniques for doubling chromosome number(Jensen, 1974; Wan et al., 1989) have been described. Colchicine is oneof the doubling agents. However, developmental abnormalities arisingfrom in vitro cloning are further enhanced by colchicine treatments, andprevious reports indicated that colchicine is toxic to microspores. Theaddition of colchicine in increasing concentrations during mannitolpretreatment prior to anther culture and microspore culture has achievedimproved percentages.

An illustrative embodiment of the combination of a chromosome doublingagent and preculture medium is one which contains colchicine. In aspecific embodiment, the colchicine level is preferably about 0.05%. Theanthers remain in the mannitol preculture medium with the additives forabout 10 days at 10° C. Anthers are then placed on maturation media, forexample, that designated 6N1-TGR-P4, for 3 to 6 weeks to induceembryoids. If the plants are to be regenerated from the embryoids, shootregeneration medium is employed, as in the isolated microspore proceduredescribed in the previous sections. Other regeneration media can be usedsequentially to complete regeneration of whole plants.

The anthers are then exposed to embryoid/callus promoting medium, forexample, that designated 6N1-TGR-P4, to obtain callus or embryoids. Theembryoids are recognized visually by identification of embryonic-likestructures. At this stage, the embryoids are transferred progressivelythrough a series of regeneration media. In an illustrative embodiment,the shoot initiation medium comprises BAP (6-benzyl-amino-purine) andNAA (naphthalene acetic acid). Regeneration protocols for isolatedmicrospore cultures and anther cultures are similar.

VIII. ADDITIONAL TISSUE CULTURES AND REGENERATION

The present invention contemplates a corn plant regenerated from atissue culture of the inbred maize plant 94INK1A, or of a hybrid maizeplant produced by crossing 94INK1A. As is well known in the art, tissueculture of corn can be used for the in vitro regeneration of a cornplant. By way of example, a process of tissue culturing and regenerationof corn is described in European Patent Application 0 160 390, thedisclosure of which is incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes (1982) andDuncan et al. (1985). The study by Duncan et al. (1985) indicates that97 percent of cultured plants produced calli capable of Iregeneratingplants. Subsequent studies have shown that both inbreds and hybridsproduced 91% regenerable calli that produced plants.

Other studies indicate that non-traditional tissues are capable ofproducing somatic embryogenesis and plant regeneration (Songstad et al.,1988; Rao et al, 1986; Conger et al, 1987; the disclosures of which areincorporated herein by reference). Regenerable cultures, including TypeI and Type II cultures, may be initiated from immature embryos usingmethods described in, for example, PCT Application WO 95/06128, thedisclosure of which is incorporated herein by reference in its entirety.

Briefly, by way of example, to regenerate a plant of this invention,cells are selected following growth in culture. Where employed, culturedcells are preferably grown either on solid supports or in the form ofliquid suspensions as set forth above. In either instance, nutrients areprovided to the cells in the form of media, and environmental conditionsare controlled. There are many types of tissue culture media comprisingamino acids, salts, sugars, hormones, and vitamins. Most of the mediaemployed to regenerate inbred and hybrid plants have some similarcomponents; the media differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but exhibit different growth rates and different morphologies, dependingon the growth media. In some media, cells survive but do not divide.Various types of media suitable for culture of plant cells have beenpreviously described and discussed above.

An exemplary embodiment for culturing recipient corn cells in suspensioncultures includes using embryogenic cells in Type II (Armstrong andGreen, 1985; Gordon-Kamm et al, 1990) callus, selecting for small (10 to30 mm) isodiametric, cytoplasmically dense cells, growing the cells insuspension cultures with hormone containing media, subculturing into aprogression of media to facilitate development of shoots and roots, andfinally, hardening the plant and readying it metabolically for growth insoil.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) can be cultured (U.S. Pat. No.5,736,369, the disclosure of which is specifically incorporated hereinby reference).

Embryogenic calli are produced essentially as described in PCTApplication WO 95/06128. Specifically, inbred plants or plants fromhybrids produced from crossing an inbred of the present invention withanother inbred are grown to flowering in a greenhouse. Explants from atleast one of the following F₁ tissues: the immature tassel tissue,intercalary meristems and leaf bases, apical meristems, immature earsand immature embryos are placed in an initiation medium which contain MSsalts, supplemented with thiamine, agar, and sucrose. Cultures areincubated in the dark at about 23° C. All culture manipulations andselections are performed with the aid of a dissecting microscope.

After about 5 to 7 days, cellular outgrowths are observed from thesurface of the explants. After about 7 to 21 days, the outgrowths aresubcultured by placing them into fresh medium of the same composition.Some of the intact immature embryo explants are placed on fresh medium.Several subcultures later (after about 2 to 3 months) enough material ispresent from explants for subdivision of these embryogenic calli intotwo or more pieces.

Callus pieces from different explants are not mixed. After furthergrowth and subculture (about 6 months after embryogenic callusinitiation), there are usually between 1 and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Material known fromexperience to lack the capacity for sustained growth is also discarded(translucent, watery, embryogenic structures). Structures with a firmconsistency resembling at least in part the scutelum of the in vivoembryo are selected.

The callus is maintained on agar-solidified MS or N6-type media. Apreferred hormone is 2,4-D. A second preferred hormone is dicamba.Visual selection of embryo-like structures is done to obtainsubcultures. Transfer of material other than that displaying embryogenicmorphology results in loss of the ability to recover whole plants fromthe callus.

Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS or N6Medium containing 2,4-D or dicamba. The calli in medium are incubated atabout 27° C. on a gyrotary shaker in the dark or in the presence of lowlight. The resultant suspension culture is transferred about once everythree to seven days, preferably every three to four days, by takingabout 5 to 10 ml of the culture and introducing this inoculum into freshmedium of the composition listed above (PCT Application WO 95/06128).

For regeneration of type I or type II callus, callus is transferred to asolidified culture medium which includes a lower concentration of 2,4-Dor other auxins than is presert in culture medium used for callusmaintenance (PCT Application WO 95/06128, specifically incorporatedherein by reference). Other hormones which can be used in regenerationmedia include dicamba, NAA, ABA, BAP, and 2-NCA. Regeneration of plantsis completed by the transfer of mature and germinating embryos to ahormone-free medium, followed by the transfer of developed plantlets tosoil and growth to maturity. Plant regeneration is described in PCTApplication WO 95/06128.

Cells from the meristem or cells fated to contribute to the meristem ofa cereal plant embryo at the early proembryo, mid proembryo, lateproembryo, transitional or early coleoptilar stage may be cultured so asto produce a proliferation of shoots or multiple meristems from whichfertile plants may be regenerated. Alternatively, cells from themeristem or cells fated to contribute to the meristem of a cereal plantimmature ear or tassel may be cultured so as to produce a proliferationof shoots or multiple meristems from which fertile plants may beregenerated (U.S. Pat. No. 5,736,369).

Progeny of any generation are produced by taking pollen and selfing,backcrossing, or sibling crossing regenerated plants by methods wellknown to those skilled in the arts. Seeds are collected from theregenerated plants. Alternatively, progeny of any generation may beproduced by pollinating a regenerated plant with its own pollen orpollen of a second maize plant. Using the methods described herein,tissue cultures and immature or mature plant tissues may be used asrecipient cell cultures for the process of genetic transformation.

IX. PROCESSES OF PREPARING CORN PLANTS AND THE CORN PLANTS PRODUCED BYSUCH CROSSES

The present invention also provides a process of preparing a novel cornplant and a corn plant produced by such a process. In accordance withsuch a process, a first parent corn plant is crossed with a secondparent corn plant wherein at least one of the first and second cornplants is the inbred corn plant 94INK1A. An important aspect of thisprocess is that it can be used for the development of novel inbredlines. For example, the inbred corn plant 94INK1A could be crossed toany second plant, and the resulting hybrid progeny each selfed for about5 to 7 or more generations, thereby providing a large number ofdistinct, pure-breeding inbred lines. These inbred lines could then becrossed with other inbred or non-inbred lines and the resulting hybridprogeny analyzed for beneficial characteristics. In this way, novelinbred lines conferring desirable characteristics could be identified.

In selecting a second plant to cross with 94INK1A for the purpose ofdeveloping novel inbred lines, it will typically be desired choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size. Alternatively, theinbred 94INK1A may be crossed with a second, different inbred plant forthe purpose of producing hybrid seed which is sold to farmers forplanting in commercial production fields. In this case, a second inbredvariety is selected which confers desirable characteristics when inhybrid combination with the first inbred line.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand.

In a preferred embodiment, crossing comprises the steps of:

(a) planting in pollinating proximity seeds of a first and a secondparent corn plant, and preferably, seeds of a first inbred corn plantand a second, distinct inbred corn plant;

(b) cultivating or growing the seeds of the first and second parent cornplants into plants that bear flowers;

(c) emasculating flowers of either the first or second parent cornplant, i.e., treating the flowers so as to prevent pollen production, oralternatively, using as the female parent a male sterile plant, therebyproviding an emasculated parent corn plant;

(d) allowing natural cross-pollination to occur between the first andsecond parent corn plants;

(e) harvesting seeds produced on the emasculated parent corn plant; and,where desired,

(f) growing the harvested seed into a corn plant, preferably, a hybridcorn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant 94INK1A is employed asthe male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof Roundup herbicide in combination with glyphosate tolerant maizeplants to produce male sterile corn plants is disclosed in U.S. patentapplication Ser. No. 08/927,368 and PCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

Alternatively, in another embodiment of the invention, both first andsecond parent corn plants can come from the same inbred corn plant,i.e., from the inbred designated 94INK1A. Thus, any corn plant producedusing a process of the present invention and inbred corn plant 94INK1A,is contemplated by the current inventor. As used herein, crossing canmean selfing, backcrossing, crossing to another or the same inbred,crossing to populations, and the like. All corn plants produced usingthe inbred corn plant 94INK1A as a parent are, therefore, within thescope of this invention.

The utility of the inbred plant 94INK1A also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Of these, Zea and Tripsacum, are most preferred. Potentiallysuitable for crosses with 94INK1A can also be the various varieties ofgrain sorghum, Sorghum bicolor (L.) Moench.

A. F₁ Hybrid Corn Plant and Seed Production

Any time the inbred corn plant 94INK1A is crossed with another,different, corn inbred, a first generation (F₁) corn hybrid plant isproduced. As such, an F₁ hybrid corn plant may be produced by crossing94INK1A with any second inbred maize plant. Therefore, any F₁ hybridcorn plant or corn seed which is produced with 94INK1A as a parent ispart of the present invention. An example of such an F₁ hybrid which hasbeen produced with 94INK1A as a parent is the hybrid 8002697. Hybrid8002697 was produced by crossing inbred corn plant 94INK1A with theinbred corn plant designated 87DIA4 (U.S. patent application Ser. No.5,936,145, the disclosure of which is specifically incorporated hereinby reference in its entirety).

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

Inbreeding requires sophisticated manipulation by human breeders. Evenin the extremely unlikely event inbreeding rather than crossbreedingoccurred in natural corn, achievement of complete inbreeding cannot beexpected in nature due to well known deleterious effects of homozygosityand the large number of generations the plant would have to breed inisolation. The reason for the breeder to create inbred plants is to havea known reservoir of genes whose gametic transmission is predictable.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by DEKALB Genetics Corporation to perform the finalevaluation of the commercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further development of theexperimental hybrids. Examples of such comparisons are presentedhereinbelow. After FACT testing is complete, determinations may be madewhether commercial development should proceed for a given hybrid.

When the inbred corn plant 94INK1A is crossed with another inbred plantto yield a hybrid, the original inbred can serve as either the maternalor paternal plant. For many crosses, the outcome is the same regardlessof the assigned sex of the parental plants.

However, there is often one of the parental plants that is preferred asthe maternal plant because of increased seed yield and productioncharacteristics. Some plants produce tighter ear husks leading to moreloss, for example due to rot. There can be delays in silk formationwhich deleteriously affect timing of the reproductive cycle for a pairof parental inbreds. Seed coat characteristics can be preferable in oneplant. Pollen can be shed better by one plant. Other variables can alsoaffect preferred sexual assignment of a particular cross. In the case ofthe instant inbred, it was generally preferable to use 94INK1A as themale parent.

B. F₁ Hybrid Comparisons

As mentioned above, hybrids are progressively eliminated followingdetailed evaluations of their phenotype, including formal comparisonswith other commercially successful hybrids. Strip trials are used tocompare the phenotypes of hybrids grown in as many environments aspossible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

Examples of such comparative data are set forth herein below in Table 2,which presents a comparison of performance data for the hybrid 8002697,a hybrid made with 94INK1A as one parent, versus selected hybrids ofcommercial value (DK405 and DK427).

All the data in Table 2 represents results across years and locationsfor research and/or strip trials. The “NTEST” represents the number ofpaired observations in designated tests at locations around the UnitedStates.

TABLE 2 Comparative Data of 8002697 SI YLD MST STL RTL DRP FLSTD SVELSTD PHT EHT BAR SG TST ESTR HYBRID NTEST % C BU PTS % % % % M RAT % MINCH INCH % RAT LBS FGDU DAYS 8002697 R 47 111.9 176.7 18.1 2.3 3.8 0.0100.5 4.9 103.1 89.9 43.8 6.5 57.9 1230 90.6 DK405 96.3 158.0 18.1 2.52.5 0.0 91.4 5.0 78.7 87.6 42.0 6.0 55.9 1214 90.2 DIFF 15.5 18.8 0.0−0.2 1.2 0.0 9.0 −0.1 24.4 2.3 1.8 0.6 2.0 16 0.4 SIG ** ** ** ** ** + +** 8002697 F 78 108.9 170.2 20.3 2.3 0.5 0.0 97.5 4.7 97.2 88.0 41.1 5.556.6 1192 91.7 DK405 106.6 166.5 18.9 4.3 0.2 0.0 102.6 3.8 104.2 85.640.6 6.4 55.5 1179 89.2 DIFF 2.3 3.7 1.4 −2.0 0.2 0.0 −5.3 0.9 −7.0 2.30.5 −0.9 1.1 13 2.5 SIG * ** * ** ** ** + * ** ** 8002697 R 47 111.9177.0 18.1 2.3 3.8 0.0 100.5 4.9 103.1 89.9 43.8 6.5 57.9 1230 90.6DK427 110.3 177.6 19.1 2.9 4.1 0.0 100.1 4.8 100.5 87.2 41.2 5.5 57.01255 91.5 DIFF 1.5 −0.7 −0.9 −0.6 −0.3 0.0 0.4 0.1 2.5 2.7 2.7 1.0 1.0−2.5 −0.9 SIG ** ** ** ** ** ** ** 8002697 F 77 105.5 164.5 19.7 3.0 1.10.0 97.6 4.8 95.8 87.6 39.3 5.8 56.7 1189 91.8 DK427 109.5 169.9 20.03.6 1.3 0.1 99.4 5.1 98.5 88.3 40.0 6.0 56.2 1220 92.3 DIFF −3.9 −5.4−0.3 −0.6 −0.3 −0.1 −1.8 −0.4 −2.7 −0.7 −0.7 −0.2 0.6 −31 −0.5 SIG *** + + + ** * Significance levels are indicated as: + = 10%, * = 5%, **= 1% LEGEND ABBREVIATIONS: HYBD = Hybrid NTEST = Research/FACT SI %C =Selection Index (percent of check) YLD BU/A = Yield (bushels/acre) MSTPTS = Moisture STL % = Stalk Lodging (percent) RTL % = Root Lodging(percent) DRP % = Dropped Ears (percent) FLSTD % M = Final Stand(percent of test mean) SV RAT = Seedling Vigor Rating ELSTD % M = EarlyStand (percent of test mean) PHT INCH = Plant Height (inches) EHT INCH =Ear Height (inches) BAR % = Barren Plants (percent) SG RAT = StaygreenRating TST LBS = Test Weight (pounds) FGDU = GDUs to Shed ESTR DAYS =Estimated Relative Maturity (days)

As can be seen in Table 2, the hybrid 8002697 differs significantly in anumber of performance traits when compared to successful commercialhybrids.

C. Physical Description of F₁ Hybrids

The present invention provides F₁ hybrid corn plants derived from thecorn plant 94INK1A. The physical characteristics of an exemplary hybridproduced using 94INK1A as one inbred parent are set forth in Table 3,which concerns 8002697. An explanation of terms used in Table 3 can befound in the Definitions, set forth herein above.

TABLE 3 Morphological Traits for the 8002697 Phenotype CHARACTERISTICVALUE 1. STALK Diameter (width) cm. 2.0 Anthocyanin Absent Nodes WithBrace Roots 1.2 Brace Root Color Faint Internode Direction StraightInternode Length cm. 16.0 2. LEAF Color Green Length cm. 81.1 Width cm.10.1 Sheath Anthocyanin Absent Sheath Pubescence Light Marginal WavesFew Longitudinal Creases Few 3. TASSEL Length cm. 45.3 Spike Length cm.23.3 Peduncle Length cm. 9.2 Branch Number 7.0 Anther Color Green-YellowGlume Color Green Glume Band Absent 4. FAR Silk Color Pink Number PerStalk 1.0 Position (attitude) Pendant Length cm. 18.3 Shape Semi-ConicalDiameter cm. 4.3 Weight gm. 174.8 Shank Length cm. 15.0 Husk Bract ShortHusk Cover cm. 2.7 Husk Opening Very Loose Husk Color Fresh Green HuskColor Dry Buff Cob Diameter cm. 2.2 Cob Color Red Shelling Percent 88.55. KERNEL Row Number 14.6 Number Per Row 41.6 Row Direction StraightType Dent Cap Color Yellow Side Color Yellow Length (depth) mm. 11.3Width mm. 8.0 Thickness 3.9 Weight of 1000K gm. 329.0 Endosperm TypeNormal Endosperm Color Yellow *These are typical values. Values may varydue to environment. Other values that are substantially equivalent arealso within the scope of the invention. Substantially equivalent refersto quantitative traits that when compared do not show statisticaldifferences of their means.

X. GENETIC COMPLEMENTS

The present invention provides a genetic complement of the inbred cornplant designated 94INK1A. Further provided by the invention is a hybridgenetic complement, wherein the complement is formed by the combinationof a haploid genetic complement from 94INK1A and another haploid geneticcomplement. Means for determining such a genetic complement arewell-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of acorn plant or a cell or tissue of that plant. By way of example, a cornplant is genotyped to determine a representative sample of the inheritedmarkers it possesses. Markers are alleles at a single locus. They arepreferably inherited in codominant fashion so that the presence of bothalleles at a diploid locus is readily detectable, and they are free ofenvironmental variation, i.e., their heritability is 1. This genotypingis preferably performed on at least one generation of the descendantplant for which the numerical value of the quantitative trait or traitsof interest are also determined. The array of single locus genotypes isexpressed as a profile of marker alleles, two at each locus. The markerallelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

A genetic marker profile of an inbred may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredof known genetic marker profile and phenotype is crossed with a secondinbred of known genetic marker profile and phenotype it is possible topredict the phenotype of the F₁ hybrid based on the combined geneticmarker profiles of the parent inbreds. Methods for prediction of hybridperformance from genetic marker data is disclosed in U.S. Pat. No.5,492,547, the disclosure of which is specifically incorporated hereinby reference in its entirety. Such predictions may be made using anysuitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

SSRs are genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present. Another advantage of this typeof marker is that, through use of flanking primers, detection of SSRscan be achieved, for example, by the polymerase chain reaction (PCR™),thereby eliminating the need for labor-intensive Southern hybridization.The PCR™ detection is done by use of two oligonucleotide primersflanking the polymorphic segment of repetitive DNA. Repeated cycles ofheat denaturation of the DNA followed by annealing of the primers totheir complementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology. Following amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size (number of base pairs) of the amplifiedsegment.

Means for performing genetic analyses using SSR polymorphisms are wellknown in the art. The SSR analyses reported herein were conducted byCelera AgGen in Davis, Calif. This service is available to the public ona contractual basis. This analysis was carried out by amplification ofsimple repeats followed by detection of marker genotypes using gelelectrophoresis. Markers were scored based on the size of the amplifiedfragment.

The SSR genetic marker profile of the parental inbreds and exemplaryresultant hybrid described herein were determined. Because an inbred isessentially homozygous at all relevant loci, an inbred should, in almostall cases, have only one allele at each locus. In contrast, a diploidgenetic marker profile of a hybrid should be the sum of those parents,e.g., if one inbred parent had the allele 168 (base pairs) at aparticular locus, and the other inbred parent had 172, the hybrid is168.172 by inference. Subsequent generations of progeny produced byselection and breeding are expected to be of genotype 168, 172, or168.172 for that locus position. When the F₁ plant is used to produce aninbred, the locus should be either 168 or 172 for that position.Surprisingly, it has been observed that in certain instances, novel SSRgenotypes arise during the breeding process. For example, a genotype of170 may be observed at a particular locus position from the cross ofparental inbreds with 168 and 172 at that locus. Such a novel SSRgenotype may further an Inbred from the parental inbreds from which itwas derived. An SSR genetic marker profile of 94INK1A and comparativeinbreds is presented in Table 4.

TABLE 4 SSR Profile of 94INK1A and Comparative Inbreds LOCUS 94INK1A311H6 94KBZ1 94INK1B BNGL105 92 92 96 92 BNGL149 183 183 165 183 BNGL252158 164 158 164 BNGL589 175 175 — 175 BNGL6l5 231 231 — 231 BNGL619 273275 — 273 DUP14 105 — 105 105 DUP28 133 133 123 133 MC1014 161 161 169161 MC1017 196 196 187 196 MC1018 138 138 138 138 MC1022 123 116 123 123MC1028 161 — 161 161 MC1043 189 189 175 189 MC1046 181 — — 181 MC1065241 — 230 241 MC1070 248 254 248 248 MC1074 164 164 174 — MC1079 173 173— 169 MC1094 178 170 178 178 MC1108 138 — — 138 MC1129 204 204 200 204MC1131 117 — 117 117 MC1138 188 190 188 188 MC1176 220 220 — 220 MC118282 82 104 82 MC1191 220 219 — — MC1194 143 143 — 143 MC1208 111 111 —111 MC1209 184 184 178 184 MC1237 159 159 159 159 MC1257 195 — 180 195MC1287 160 160 160 160 MC1288 113 113 — 113 MC1305 160 200 160 160MC1325 171 171 179 179 MC1329 95 — 109 95 MC1360 143 143 151 143 MC1429199 191 199 199 MC1449 97 95 96 — MC1484 117 124 117 117 MC1520 283 275284 275 MC1523 199 199 200 200 MC1538 237 237 223 237 MC1605 134 110 —134 MC1662 161 161 136 161 MC1720 245 245 — 245 MC1732 102 100 102 102MC1740 167 167 129 167 MC1782 228 228 228 228 MC1784 254 254 248 254MC1808 147 131 147 147 MC1831 178 184 186 178 MC1834 208 208 207 208MC1839 194 194 — 194 MC1866 133 123 133 133 MC1890 185 — 167 195 MC1904193 191 191 183 MC1917 169 169 141 169 MC1931 174 — — 174 MC2047 144 144144 144 MC2086 229 247 229 229 MC2132 223 223 223 223 MC2238 191 195 191191 MC2259 180 180 — 180 MC2305 190 216 180 190 NC004 148 148 185 185PHI017 110 — 113 110 PHI024 177 — 177 177 PHI031 198 198 194 198 PHI033257 257 257 257 PHI037 139 139 137 139 PHI050 92 — 90 92 PHI051 149 149149 149 PHI061 85 85 — 85 PHI064 90 90 90 90 PHI065 138 — 138 138 PHI072149 149 — 149 PHI078 133 133 129 129 PHI089 92 92 93 92 PHI093 293 293293 293 PHI101 99 99 — 99 PHI116 181 177 177 — PHI119 168 168 168 168PHI120 76 — — 76 Primers used to detect SSRs are from Celera AgGen,Inc., 1756 Picasso Avenue, Davis, CA, 95616

Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. Thetechniques and nomenclature for isozyme analysis are described in, forexample, Stuber et al. (1988), which is incorporated by reference.

A standard set of loci can be used as a reference set. Comparativeanalysis of these loci is used to compare the purity of hybrid seeds, toassess the increased variability in hybrids compared to inbreds, and todetermine the identity of seeds, plants, and plant parts. In thisrespect, an isozyme reference set can be used to develop genotypic“fingerprints.”

Table 5 lists the identifying numbers of the alleles at isozyme locitypes, and represents the exemplary genetic isozyme typing profile for94INK1A.

TABLE 5 Isozyme Profile of 94INK1A and Comparative Inbreds ISOZYMEALLELE LOCUS 94INK1A 311H6 94KBZ1 94INK1B Acph1 2 2 3 2 Adh1 — 4 4 4Cat3 — 9 9 — Got3 — 4 4 — Got2 — 4 4 — Got1 — — 4 — Idh1 4 4 4 4 Idh2 66 4 6 Mdh1 6 6 6 6 Mdh2 3.5 3.5 6 3.5 Mdh3 16 16 16 16 Mdh4 12 12 12 12Mdh5 12 12 12 12 Pgm1 9 9 9 9 Pgm2 4 4 4 4 6Pgd1 3.8 3.8 2 3.8 6Pgd2 5 55 5 Phi1 — 4 4 4

The present invention also provides a hybrid genetic complement formedby the combination of a haploid genetic complement of the corn plant94INK1A with a haploid genetic complement of a second corn plant. Meansfor combining a haploid genetic complement from the foregoing inbredwith another haploid genetic complement can comprise any method forproducing a hybrid plant from 94INK1A. It is contemplated that such ahybrid genetic complement can be prepared using in vitro regeneration ofa tissue culture of a hybrid plant of this invention.

A hybrid genetic complement contained in the seed of a hybrid derivedfrom 94INK1A is a further aspect of this invention. An exemplary hybridgenetic complement is that of the hybrid 8002697.

Table 6 shows the identifying numbers of the alleles for the hybrid8002697, which constitutes an exemplary SSR genetic marker profile forhybrids derived from the inbred of the present invention. Table 6concerns 8002697, which has 94INK1A as one inbred parent.

TABLE 6 SSR Profile of 8002697 LOCUS Hybrid 8002697 BNGL149 183.187BNGL252 158.166 BNGL589 175.175 BNGL619 273.265 DUP14 105.112 DUP28133.125 MC1014 161.161 MC1017 196.196 MC1018 138.130 MC1022 123.67MC1028 161.159 MC1043 189.175 MC1046 181.218 MC1065 241.230 MC1070248.239 MC1074 164.186 MC1079 173.177 MC1094 178.182 MC1108 138.122MC1129 204.206 MC1131 117.109 MC1138 188.186 MC1176 220.218 MC118282.106 MC1191 220.203 MC1194 143.143 MC1208 111.111 MC1209 184.184MC1237 159.159 MC1257 195.187 MC1287 160.160 MC1288 113.117 MC1305160.160 MC1325 171.175 MC1329 95.107 MC1360 143.133 MC1429 199.191MC1449 97.96 MC1484 117.124 MC1520 283.283 MC1523 199.200 MC1538 237.237MC1605 134.110 MC1662 161.169 MC1720 245.241 MC1732 102.100 MC1740167.161 MC1782 228.228 MC1784 254.250 MC1808 147.137 MC1831 178.186MC1834 208.216 MC1839 194.184 MC1866 133.119 MC1890 195.136 MC1904193.187 MC1917 169.109 MC1931 174.173 MC2047 144.146 MC2086 229.242MC2132 223.223 MC2259 180.173 MC2305 190.180 NC004 148.148 PHI024177.171 PHI031 198.194 PHI033 257.257 PHI037 139.141 PHI050 92.92 PHI051149.149 PHI061 85.93 PHI064 90.104 PHI065 138.158 PHI072 149.150 PHI078133.129 PHI089 92.93 PHI093 293.299 PHI101 99.103 PHI116 181.181 PHI119168.172 PHI120 76.73 Primers used to detect SSRs are from Celera AgGen,Inc., 1756 Picasso Avenue, Davis, CA, 95616

The exemplary hybrid genetic complements of hybrid 8002697 may also beassessed by genetic isozymne typing profiles using a standard set ofloci as a reference set, using, e.g., the same, or a different, set ofloci to those described above. Table 7 lists the identifying numbers ofthe alleles at isozyme loci types and presents the exemplary geneticisozyme typing profile for the hybrid 8002697, which is an exemplaryhybrid derived from the inbred of the present invention. Table 7concerns 8002697, which has 94INK1A as one inbred parent.

TABLE 7 Isozyme Profile for Hybrid 8002697 Locus Isozyme Allele Acph1 2Adh1 4 Cat3 9 Got3 4 Got2 4 Got1 4 Idh1 4 Idh2 6 Mdh1 6 Mdh2 3.5 Mdh3 16Mdh4 12 Mdh5 12 Pgm1 9 Pgm2 4 6-Pgd1 3.8 6-Pgd2 5 Phi1 4

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Anderson, W. P., Weed Science Principles, West Publishing Company, 1983.

Armstrong and Green, “Establishment and maintenance of friable,embryogenic maize callus and the involvement of L-proline,” Planta,164:207-214, 1985.

Conger, Novak, Afza, Erdelsky, “Somatic Embryogenesis from Cultured LeafSegments of Zea Mays,” Plant Cell Reports, 6:345-347, 1987.

Duncan et al., “The Production of Callus Capable of Plant Regenerationfrom Immature Embryos of Numerous Zea Mays Genotypes,” Planta,165:322-332, 1985.

Fehr, “Theory and Technique,” In: Principles of Cultivar Development,1:360-376, 1987.

Gaillard et al., “Optimization of Maize Microspore Isolation and CultureCondition for Reliable Plant Regeneration,” Plant Cell Reports,10(2):55, 1991.

Gordon-Kamm et al., “Transformation of Maize Cells and Regeneration ofFertile Transgenic Plants,” The Plant Cell, 2:603-618, 1990.

Green and Rhodes, “Plant Regeneration in Tissue Cultures of Maize:Callus Formation from Stem Protoplasts of Corn (Zea Mays L.),” In: Maizefor Biological Research, 367-372, 1982.

Jensen, “Chromosome Doubling Techniques in Haploids,” Haploids andHigher Plants—Advances and Potentials, Proceedings of the FirstInternational Symposium, 1974.

Nienhuis et al., “Restriction Fragment Length Polymorphism Analysis ofLoci Associated with Insect Resistance in Tomato,” Crop Science,27(4):797-803, 1987.

Omirulleh et al., “Activity of a chimeric promoter with the doubled CaMV35S enhancer element in protoplast-derived cells and transgenic plantsin maize,” Plant Mol. Biol., 21(3):415-428, 1993.

Pace et al., “Anther Culture of Maize and the Visualization ofEmbryogenic Microspores by Fluorescent Microscopy,” Theoretical andApplied Genetics, 73:863-869, 1987.

Poehlman et al., “Breeding Field Crops,” 4th Ed., Iowa State UniversityPress, Ames, IA, pp 132-155 and 321-344, 1995.

Rao et al., “Somatic Embryogenesis in Glume Callus Cultures,” MaizeGenetics Cooperation Newsletter, 60, 1986.

Songstad et al., “Effect of 1-Aminocyclopropate-1-Carboxylic Acid,Silver Nitrate, and Norbornadiene on Plant Regeneration from MaizeCallus Cultures,” Plant Cell Reports, 7:262-265, 1988.

Sprague and Dudley (eds.), “Corn and Corn Improvement,” 3rd Ed., CropScience of America, Inc., and Soil Science of America, Inc., MadisonWis. pp 881-883 and pp 901-918, 1988.

Stuber et al., “Techniques and scoring procedures for starch gelelectrophoresis of enzymes of maize C. Zea mays, L.,” Tech. Bull., 286,1988.

Wan et al., “Efficient Production of Doubled Haploid Plants ThroughColchicine Treatment of Anther-Derived Maize Callus,” Theoretical andApplied Genetics, 77:889-892, 1989.

Wang et al., “Large-Scale Identification, Mapping, and Genotyping ofSingle-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082, 1998.

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What is claimed is:
 1. Inbred corn seed of the corn plant 94INK1A, asample of said seed having been deposited under ATCC Accession No.PTA-2119.
 2. The inbred corn seed of claim 1, further defined as anessentially homogeneous population of inbred corn seed.
 3. The inbredcorn seed of claim 1, further defined as essentially free from hybridseed.
 4. An inbred corn plant produced by growing the seed of the inbredcorn plant 94INK1A, a sample of said seed having been deposited underATCC Accession No. PTA-2119.
 5. Pollen of the plant of claim
 4. 6. Anovule of the plant of claim
 4. 7. An essentially homogeneous populationof corn plants produced by growing the seed of the inbred corn plant94INK1A, a sample of said seed having been deposited under ATCCAccession No. PTA-2119.
 8. A corn plant capable of expressing all thephysiological and morphological characteristics of the inbred corn plant94INK1A, a sample of the seed of said inbred corn plant 94INK1A havingbeen deposited under ATCC Accession No. PTA-2119.
 9. The corn plant ofclaim 8, further comprising a factor conferring male sterility.
 10. Atissue culture of regenerable cells of inbred corn plant 94INK1A,wherein the tissue regenerates plants capable of expressing all thephysiological and morphological characteristics of the inbred corn plant94INK1A, a sample of the seed of said inbred corn plant 94INK1A havingbeen deposited under ATCC Accession No. PTA-2119.
 11. The tissue cultureof claim 10, wherein the regenerable cells comprise cells derived fromembryos, immature embryos, meristematic cells, immature tassels,microspores, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.
 12. The tissue culture of claim11, wherein the regenerable cells comprise protoplasts or callus.
 13. Acorn plant regenerated from the tissue culture of claim 10, wherein saidcorn plant is capable of expressing all of the physiological andmorphological characteristics of the inbred corn plant designated94INK1A, a sample of the seed of said inbred corn plant designated94INK1A having been deposited under ATCC Accession No. PTA-2119.
 14. Aninbred corn plant cell of the corn plant of claim 8, said cellcomprising: (a) an SSR genetic marker profile in accordance with theprofile shown in Table 4; or (b) a genetic isozyme typing profile inaccordance with the profile shown in Table
 5. 15. A corn seed comprisingthe inbred corn plant cell of claim
 14. 16. A tissue culture comprisingthe inbred corn plant cell of claim
 14. 17. The inbred corn plant ofclaim 8, comprising: (a) an SSR genetic marker profile in accordancewith the profile shown in Table 4; or (b) a genetic isozyme typingprofile in accordance with the profile shown in Table
 5. 18. A processof producing corn seed, comprising crossing a first parent corn plantwith a second parent corn plant, wherein said first or second corn plantis the inbred corn plant 94INK1A, a sample of the seed of said inbredcorn plant 94INK1A having been deposited under ATCC Accession No.PTA-2119, wherein seed is allowed to form.
 19. The process of claim 18,further defined as a process of producing hybrid corn seed, comprisingcrossing a first inbred corn plant with a second, distinct inbred cornplant, wherein said first or second inbred corn plant is the inbred cornplant 94INK1A, a sample of the seed of said inbred corn plant 94INK1Ahaving been deposited under ATCC Accession No. PTA-2119.
 20. The processof claim 19, wherein crossing comprises the steps of: (a) planting inpollinating proximity seeds of said first and second inbred corn plants;(b) cultivating the seeds of said first and second inbred corn plantsinto plants that bear flowers; (c) emasculating the male flowers of saidfirst or second inbred corn plant to produce an emasculated corn plant;(d) allowing cross-pollination to occur between said first and secondinbred corn plants; and (e) harvesting seeds produced on saidemasculated corn plant.
 21. The process of claim 20, further comprisinggrowing said harvested seed to produce a hybrid corn plant.
 22. Hybridcorn seed produced by the process of claim
 20. 23. A hybrid corn plantproduced by the process of claim
 21. 24. The hybrid corn plant of claim23, wherein the plant is a first generation (F₁) hybrid corn plant. 25.The corn plant of claim 4, further comprising a single locus conversion.26. The corn plant of claim 25, wherein the single locus was stablyinserted into a corn genome by transformation.
 27. The corn plant ofclaim 25, wherein the locus is selected from the group consisting of adominant allele and a recessive allele.
 28. The corn plant of claim 25,wherein the locus confers a trait selected from the group consisting ofherbicide resistance; insect resistance; resistance to bacterial,fungal, nematode or viral disease; yield enhancement; waxy starch;improved nutritional quality; enhanced yield stability; male sterilityand restoration of male fertility.